Bioreactor and related methods

ABSTRACT

An apparatus for culturing cells includes a bioreactor. The bioreactor may be modular and may include in a chamber a fixed bed, such as an unstructured or structured fixed bed (such as a spiral bed) for culturing cells, with a return column arranged centrally within the chamber. The modular bioreactor may include a plurality of structured fixed bed arranged in a stacked configuration. The modular bioreactor may include an outer casing forming a space for conditioning (e.g., insulating, heating, cooling) at least a chamber in which cells are cultured. The bioreactor may also include an impeller with radially curved blades, and may also suspend the impeller so that it may move from side-to-side and align with an external drive. Related methods are also disclosed.

This application is a continuation of U.S. Utility application Ser. No.17/887,898 filed on Aug. 15, 2022 which is a continuation of U.S.application Ser. No. 16/955,904 filed on Jun. 19, 2020, which is anational stage of International Patent Application PCT/EP2018/086394,which claims the benefit of U.S. Provisional Application Ser. Nos.62/758,152, 62/733,375 and 62/608,261, all of which are incorporatedherein by reference.

TECHNICAL FIELD

This document relates generally to the cell culturing arts and, moreparticularly, to a bioreactor and related methods.

SUMMARY

An objective of this disclosure is to provide a bioreactor, which may bein modular form, that utilizes one or more structured fixed beds topromote case of manufacturing and use, while still achieving excellentcell culturing outcomes from the resulting homogeneity and repeatabilityafforded, even when scaled up or down.

According to a first aspect of the disclosure, an apparatus forculturing cells in connection with a fluid flow, comprising a modularbioreactor including a fixed bed for culturing cells.

In some embodiments, the modular bioreactor comprises a base portionhaving a first chamber, an intermediate portion forming at least part ofa second, outer chamber for receiving the fixed bed and at least part ofa third inner chamber for returning fluid flow from the second outerchamber to the first chamber, and a cover portion for positioning overthe intermediate portion. The fixed bed may comprise a structured fixedbed, and the intermediate portion may comprise a tubular part, thestructured fixed bed extending spirally around the tubular part, or theintermediate portion may comprise an inner wall of the fixed bed. In anyembodiment, the intermediate portion may comprise a plurality ofintermediate parts, each associated with a structured fixed bed.

In some embodiments, at least one of the plurality of intermediate partsis perforated for allowing fluid to flow from a first structured fixedbed below the at least one intermediate part to a second structuredfixed bed above the at least one intermediate part. In some embodiments,each of the plurality of intermediate parts is tubular, and eachstructured fixed bed comprises a spiral bed wound around the tubularintermediate part. A perforated support may be provided for thestructured fixed bed.

In some embodiments, the intermediate portion may further comprise atubular casing for forming a periphery of the modular bioreactor. Thetubular casing forms a space for heating, cooling, or insulating thebioreactor. The intermediate portion may comprise a plurality ofintermediate parts, each adapted for connecting with each other.

In some embodiments, the intermediate portion includes a tube forengaging at least one intermediate part and forming an inner wall of theouter second chamber for receiving the fixed bed. The tub may engagewherein the tube engages a first intermediate part below the tube and asecond intermediate part above the tube. The second intermediate partmay include openings for creating a fluid film along the third innerchamber. Supports, such as vertical rods, may be provided for supportingthe second intermediate part from the first intermediate part.

In some embodiments, the cover portion comprises a removable capincluding a plurality of ports. The removable cap may have an outerdiameter that is less than an outer diameter of the intermediateportion. At least one of the ports may include a threaded metal insert.The cover portion may have an outer diameter that is equal to or greaterthan an outer diameter of the intermediate portion.

The intermediate portion may comprise an intermediate part adapted forpositioning at least partially within the base portion. The intermediatepart may further include a flow disruptor for disrupting fluid flow.

The base portion may include a further chamber radially outward of thefirst chamber in fluid communication with the second outer chamberincluding the fixed bed. This further chamber may be formed by anupstanding wall having a plurality of openings for transmitting fluidfrom the first chamber to the further chamber.

In some embodiments, an agitator is associated with the base portion.The intermediate portion may be adapted for suspending the agitator inthe first chamber in a manner that allows side-to-side movement foralignment with an external drive.

In some embodiments, a container is provided for containing theagitator, the container including a central inlet and a plurality ofradially oriented outlets. A flow divider may be associated with thecentral inlet. In any embodiment, or as an independent componentseparate from any bioreactor, the agitator may comprise a plurality ofcurved blades.

In some embodiments, a plurality of flow disruptors are provided fordividing the fluid flow entering the third inner chamber into aplurality of streams. The plurality of flow disruptors may be associatedwith a ring. In some embodiments, one or more conduits for permittinggas to enter into a space behind one of the streams. The one or moreconduits may be connected to a structure including the plurality of flowdisruptors. For example, a first conduit may be connected to thestructure, or both first and second conduits may be connected to thestructure. Alternatively, the first and second conduits may not beconnected to the structure.

According to a further aspect of the disclosure, an apparatus forculturing cells is disclosed. The apparatus comprises a modularbioreactor comprising a base portion removably connected to both acentral column and an outer casing, the outer casing and central columntogether forming a compartment for culturing cells.

In some embodiments, the compartment includes at least one structuredfixed bed. The compartment may include a plurality of structured fixedbeds, arranged in a stacked configuration. An intermediate part, such asa screen, may be positioned between at least two of the plurality ofstructured fixed beds.

In some embodiments, the at least one structured fixed bed comprises aspiral bed. Each of the plurality of stacked, structured fixed beds iswrapped around the central column. The central column comprises firstand second interconnected tubes, a first structured fixed bed of theplurality of structured fixed beds being wrapped around the first tubeand a second structured fixed bed of the plurality of structured fixedbeds being wrapped around the second tube. The central column comprisesfirst and second tubes for engaging a perforated support extendingbetween at least two of the plurality of structured fixed beds.

In any embodiment, the structured fixed bed may comprise a cartridgeadapted for being inserted into and removed from the second, outerchamber or compartment.

According to a further aspect of the disclosure, a bioreactor forculturing cells is provided. The bioreactor may comprise a base parthaving a first chamber including an agitator for agitating a fluid. Afirst central column may be attached to the base part, optionallyremovably, the first central column forming at least part of a second,outer chamber for culturing cells and a third inner chamber forreturning fluid flow from the second outer chamber to the first chamber.

In this or other embodiments, the second, outer chamber includes a firststructured fixed bed. In this or any embodiment, the first structuredfixed bed comprises a spiral bed, and may be wound or wrapped around thefirst central column. A second central column may also form at leastpart of the second outer chamber, and further including a secondstructured fixed bed spaced vertically from the first structured fixedbed. A perforated support may be provided between the first structuredfixed bed and the second structured fixed bed.

In any embodiment, the second, outer chamber includes an unstructuredbed.

According to yet another aspect of the disclosure, a bioreactor forculturing cells in connection with a fluid is disclosed. The bioreactorcomprises a first chamber including an agitator for agitating the fluid,a second, outer chamber including a plurality of stacked beds forculturing cells, and a third, inner chamber for returning fluid from thesecond outer chamber to the first chamber.

In some embodiments, the bioreactor comprises a base portion having thefirst chamber, an intermediate portion forming at least part of thesecond, outer chamber and at least part of the third inner chamber, anda cover portion for positioning over the intermediate portion. In thisor other embodiments, the intermediate portion comprises a first supportfor supporting a first bed of the plurality of stacked beds. Theintermediate portion comprises a second support for supporting a secondbed of the plurality of stacked beds, and may be adapted for removablyconnecting with the base portion and the cover portion.

In some embodiments, the second, outer chamber is bounded by an outerwall. The bioreactor may further include an outer casing forming a spacewith the outer wall, the space being for insulating, heating, or coolingthe second, outer chamber.

Still another aspect of the disclosure pertains to a bioreactor forculturing cells in connection with a fluid. The bioreactor comprises afirst chamber including an agitator for agitating the fluid, a second,outer chamber including at least one bed for culturing cells, and athird, inner chamber for returning fluid from the second outer chamberto the first chamber. The second, outer chamber may be bounded by anouter wall, and further including an outer casing forming a space withthe outer wall, the space being for insulating, heating, or cooling thesecond, outer chamber.

In this or any other embodiments, the at least one bed comprises astructured fixed bed, such as a spiral bed, but could also be anunstructured bed. The inner chamber may be formed by at least one tube(which may be separate from or part of the bed). The least one tube maybe connected to first and second supports bounding the at least one bed.The first and second supports may be connected to the outer wall, or thefirst and second supports may be at least partially perforated.

Yet another portion of the disclosure pertains to an apparatus forculturing cells, comprising a bioreactor including an agitator, thebioreactor adapted for maintaining the agitator in a suspended conditionthat allows side-to-side movement for alignment with an external drive.

In some embodiments, the bioreactor includes a base portion forreceiving the agitator, and an intermediate portion for supporting acarrier for carrying the agitator in the suspended condition. Thecarrier may comprise a clip for engaging the intermediate portion.

A further aspect of the disclosure relates to an apparatus for culturingcells. The apparatus comprises a bioreactor including an agitator havinga plurality of curved blades. The agitator may include a central openregion radially inward of the plurality of curved blades, and mayinclude one or more magnets.

This disclosure also relates to bioreactor comprising first and secondstacked, structured beds. The bioreactor may further include a screenengaging both the first and second stacked, structured beds. The firstand second stacked beds may comprise structured beds, such as spiralbeds.

Also disclosed is a bioreactor including a structured fixed bed forminga central column of the bioreactor. The structured fixed bed maycomprise a spiral bed. An inner surface of the structured fixed bed isfluid-impervious, such that a central column is formed for returningfluid to recirculation through the structured fixed bed, such as fromtop to bottom. The bioreactor may be modular, and a plurality ofstacked, structured fixed beds may be provided, possibly with a gap orspacer between each bed in the stack.

According to still a further aspect of the disclosure, a method ofmanufacturing a bioreactor is provided. The method includes connecting abase portion including a first chamber with an agitator for agitating afluid to at least one intermediate portion forming at least portion of asecond, outer chamber for culturing cells in connection with fluidtransferred from the second, outer chamber, and a third, inner chamberfor returning fluid to the first chamber of the base portion.

In some embodiments, the method includes the step of connecting a coverportion over the at least one intermediate portion. The method mayfurther include the step of spirally wrapping a matrix material aroundthe intermediate portion to form a structured fixed bed for culturingcells in the outer chamber, or inserting a structured fixed bed into thesecond, outer chamber. The method may further include providing an outercasing to form a periphery of the second, outer chamber, or connectingthe outer casing to the base portion. A further step involves stacking aplurality of structured fixed beds in the second, outer chamber, orproviding a perforated support between the plurality of structured fixedbeds. In any embodiment, the method may include the step of suspendingthe agitator above the base portion in a manner that permitsside-to-side movement to align with an external drive.

Yet another aspect of the disclosure is a method of manufacturing abioreactor, comprising providing a plurality of structured fixed beds inthe bioreactor. The method may further include the step of providing aperforated spacer between each of the plurality of structured fixedbeds. The method may further include the step of providing an inner tubealong an inner side of each structured fixed bed and an outer tube alongan outer side of each structured fixed bed. Still further, the methodmay include the step of providing a casing radially outward of the outertube, the casing creating a space for insulating, heating, or coolingthe bioreactor.

BRIEF DESCRIPTION OF THE DRAWING FIGURES

The novel features of the disclosure are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present disclosure will be obtained by reference tothe following detailed description that sets forth illustrativeembodiments, in which the principles of the invention are utilized, andthe accompanying drawings of which:

FIG. 1 is a perspective view of a first embodiment of a bioreactoraccording to the disclosure.

FIGS. 2A, 2B, and 2C illustrate a possible environment of use of thebioreactor of FIG. 1 .

FIG. 3 is a perspective view of the bioreactor of FIG. 1 , includingseveral enlarged views.

FIGS. 3A, 3B and 3C illustrate a matrix material for use in forming astructured fixed bed for culturing cells in any of the disclosedbioreactors.

FIG. 4 illustrates a modular version of the bioreactor of FIG. 1 .

FIG. 5 is a cross-sectional view of a second embodiment of a bioreactoraccording to the disclosure.

FIG. 6 is a cross-sectional view of a base portion of the bioreactor ofFIG. 5 .

FIG. 7 is a partially cutaway top view of an intermediate part of thebioreactor of FIG. 5 .

FIG. 8 is a partially cutaway bottom view of an intermediate part of thebioreactor of FIG. 5 .

FIG. 9 is a perspective view of a cover portion of the bioreactor ofFIG. 5 .

FIG. 10 is a cross-sectional view of a manner of providing metal threadsin a plastic port.

FIGS. 11, 11A and 11B are various view of a third embodiment of abioreactor according to the disclosure.

FIG. 12 is a cross-sectional view of the bioreactor of FIG. 11 .

FIG. 13 is cross-sectional view of the bioreactor of FIG. 11 .

FIG. 14 is a cross-sectional view of a fourth embodiment of a bioreactoraccording to the disclosure.

FIG. 15 is a partially cutaway view of a portion of the bioreactor ofFIG. 14 .

FIG. 16 is a partially cutaway view of portion of the bioreactor of FIG.14 .

FIGS. 16A, 16B, and 16C are a cross-sectional views of furtherembodiments of the bioreactor of FIG. 14 .

FIGS. 17 and 18 are schematic views of a fifth embodiment of abioreactor according to the disclosure.

FIGS. 19 and 20 are bottom and top views of an embodiment of animpeller.

FIG. 21 is an illustration of various forms of impellers and associatedhousings.

FIG. 22 is top view of another impeller according to the disclosure.

FIGS. 23 and 24 illustrate an embodiment of a flow disruptor.

FIGS. 25 and 26 illustrate the use of conduits for supplying a gas to aportion below a “waterfall” of a bioreactor.

FIGS. 27 and 28 illustrate embodiments of a probe for use in connectionwith a bioreactor.

FIGS. 29 and 30 are graphs illustrating testing of the bioreactor.

FIGS. 31 and 32 are graphs illustrating testing of the bioreactor toassess cell density of structured fixed beds in a stacked configuration.

FIGS. 33, 34, 35, 36, 37, and 38 schematically illustrate a method ofmanufacturing a modular bioreactor according to the disclosure.

DETAILED DESCRIPTION

Bioreactors are frequently used for culturing cells. Typical bioreactorsare constructed with fixed dimensions (height, in particular), and thuscan be difficult and costly to transport to remote locations where cellcultures may be needed for providing treatments (particularly, thedeveloping world). The fixed nature also prevents past bioreactors frombeing adapted for a variety of uses.

A further issue relates to the ability of maximizing cell density for agiven area. Many past proposals for bioreactors use fluidized beds.While such beds may work well for promoting cell growth and providecertain advantages, the resulting volume of space in the bioreactorrequired to create such a bed is large. Readily scaling a bioreactorwith an unstructured or fluidized bed while achieving the desired cellgrowth is also challenging, and there is a current demand forbioreactors that may be utilized in a variety of operating conditions inthe field (including, for example, within a sterile hood, whereclearance may be limited).

Accordingly, a need is identified for an improved bioreactor that wouldbe easy to deliver and assemble, particularly at remote locations,and/or would be readily adaptable for use in a variety of sizes orconfigurations, or for different applications or uses. In someembodiments of the disclosure, the present disclosure concerns systemsand methods for the production of biologics. In particular, theproduction of cells, viruses or cells- or virus-derived products.

In some embodiments, a bioreactor disclosed herein allows for highdensity cell growth. For example, density of at least 2 millioncells/ml, at least 5 million cells/ml, at least 10 million cells/ml, atleast 20 million cells/ml, at least 40 million cells/ml, at least 60million cells/ml, or at least 100 million cells/ml. In some embodiments,the density can reach 300, 250 or 200 million cells/ml. In someembodiments, the bioreactor disclosed herein can have a total volume ofat least 1 L, at least 10 L, at least 30 L, at least 40 L, or at least50 L. In some embodiments, the bioreactor total volume can be at most2500 L, at most 200 L, at most 150 L, at most 100 L, or at most 75 L. Bybioreactor total volume reference can be made to the total liquid volumethat can be introduced in the bioreactor, which will then be full.

In some embodiments, the bioreactor can be a perfusion bioreactor, wavebioreactor, cylindrical bioreactor, bag bioreactor, moving bedbioreactor, packed bed bioreactor, fibrous bioreactor, membranebioreactor, batch bioreactor, or continuous bioreactor. In someembodiments, the bioreactors can be made from or comprise a suitablematerial, for example, stainless steel, glass, aluminum, or plastic. Insome embodiments, the bioreactor can allow for downstream analysis ofproducts. In some embodiments, a bioreactor described herein can beconnected with or to one or more inactivation units to inactivate forexample a produced product (e.g. virus), a concentrator, or apurification unit. In some embodiments, a concentrator is a devicesuited for reducing the volume of the liquid in which target biomoleculeresides. In some embodiment, the concentrator comprises a tangentialflow filter or a dead-end filter. In some embodiment, the concentratoris based on filtration and/or size exclusion chromatograph. In someembodiments, the concentrator can be a filtration device, amicro-filtration device, or an ultra-filtration device or a combinationof both micro- and ultra-filtration device. In some embodiments, apurification unit described herein can comprise a filtration device, anultrafiltration device, a difiltration device, a pH adjustment device, acentrifugation device, a washing device, a chromatography column (e.g.affinity chromatography, ionic exchange chromatography, hydrophobicinteraction chromatography, size exclusion chromatography, orimmune-affinity chromatography), a chromatography membrane, a harvestdevice, a dialysis device, a concentration device, or any combinationthereof.

Access to a bioreactor described herein can be via a lid, or door. Insome embodiments, an access mechanism for the bioreactor can comprisefor example, a lock and key mechanism, a pass code punch pad, cardswipe, transponder reader, finger print scanner, retina scanner,sensors, automatic identification and data capture methods such asradio-frequency identification (RFID), biometrics (like iris or facialrecognition system), magnetic stripes, Optical character recognition(OCR), smart cards, voice recognition, or any other access mechanism.

In some embodiment, a bioreactor disclosed herein can comprise a processcontroller. In some embodiments, the process controller is configured tocontrol operations of a bioreactor and can include a plurality ofsensors, a local computer, a local server, a remote computer, a remoteserver, or a network. In some embodiments, the bioreactor can includeone or more sensors, for example, a temperature sensor (e.g., athermocouple), flow rate sensor, gas sensor, or any other sensor. Insome embodiments, the process controller can be operational to controlaspects of a product manufacturing process, and can be coupled tosensors disposed in the bioreactor, for example, to control thetemperature, volume flow rate or gas flow rate into the bioreactor inreal time. In some embodiments, a process controller can include adisplay, for example, a computer monitor, a smart phone app, a tabletapp, or an analog display, that can be accessed by a user to determinethe state of the system. In some embodiments, the process controller caninclude an input, for example, a keyboard, a key pad, a mouse, or atouch screen, to allow a user to enter control parameters forcontrolling the operation of the bioreactor. In some embodiments, theprocess controller can control access to the bioreactor.

In some embodiments, the bioreactor disclosed herein can comprise and orcontain sensors for monitoring different parameters. In someembodiments, a sensor disclosed herein can be located in any compartmentof a bioreactor disclosed herein. In some embodiment, sensors describedherein can be a gas sensor (e.g. oxygen, nitrogen, or carbon dioxide),pH sensor, temperature sensor, cell density sensor, or dissolved oxygensensor. In some embodiments, the sensors disclosed herein can measureamongst other things, biomass or cell density, the dissolved oxygenpartial pressure, oxygen content, the ρTI value, the temperature,certain concentrations of nutriments, such as lactate, ammonium,carbonates, glucose or any metabolic product or product to bemetabolized which could for example reflect the cell density. In someembodiment, cell density (biomass density) can be determined byelectrical impedance analysis or electrical impedance spectroscopy usingan arrangement of measuring electrode. In some embodiments, a bioreactoraccording to the disclosure can comprise sensors for measuring cultureparameters. In some embodiments, a sensor disclosed herein can be incontact with culture medium in the bioreactor. In some embodiments,culture parameters can comprise amongst other things, the dissolvedoxygen partial pressure, the pH, the temperature, the optical density,certain concentrations of nutriments, such as lactate, ammonium,carbonates, glucose or any metabolic product or product to bemetabolized which could for example reflect the cell density. In someembodiment, a bioreactor disclosed herein can use regulation loopsaccording to the disclosed parameters. In some embodiments, a regulationloop can for example, modulate the quantity of oxygen to be injectedaccording to the value of the dissolved oxygen partial pressure presentor the quantity of dissolved oxygen consumed by the cells; speed ofcirculation of the culture medium; inject CO₂ according to the pH valueobtained by the sensors or any other type of regulation generally usedin this type of culture. In some embodiments, cells can be exposed todissolved oxygen concentrations of 300 μM or less (160 mmHg partialpressure), less than 200 μM, or between 20 and 150 μM. In someembodiments, cells can be exposed to about 0%, 1%, 5%, 10%, 20%, 30%,40%, 50%, 60%, 70%, 78%, 80%, 90%, or 100% nitrogen and/or about 0%, 1%,5%, 10%, 21%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% oxygen. In someembodiments, cells can be exposed to pure oxygen or an oxygen enrichedatmosphere.

In some embodiments, a bioreactor disclosed herein may comprise heatingand/or cooling devices, designed to heat and/or cool culture medium. Insome embodiments, the heating device can be an electrical element, anelectrical coil or any other heating means generally used in the fieldof cell culture, such as for example a thermostatically controlleddouble jacket. In some embodiments, cooling device may be any suitablecooling devices such as a Peltier element. In some embodiments, withregard to the culture medium and gas, the bioreactor comprises at leastone inlet for the introduction of gas and/or culture medium and at leastone outlet for the collection of the culture medium contained in thebioreactor. In some embodiments, mix of gas or gaseous mixture andculture medium can be supplied to through the same supply line.

In some embodiments, culture medium can be circulated via an agitator.In some embodiments, and agitator can be a rotatable, non-contactmagnetic impeller, a blade or screw agitation system, or an externalcirculation system. In some embodiments, the agitator can comprise adisk blade turbine, a curved blade turbine, an open lade fluid foilaxial impeller, a turbine impeller with pitched blades, or a three-bladepropeller. In some embodiments, the agitator can have a flow rate ofless than about 0.01 l/min, 0.05 l/min, 0.1 l/min, 0.5 l/min, 1 l/min, 2l/min, 5 l/min, 10 l/min, 15 l/min, 20 l/min, 50 l/min, 100 l/min, or150 l/min to more than about 160 l/min, 180 l/min, 200 l/min, or 250l/min.

In some embodiments, the bioreactor described herein comprises a fixedbed. In some embodiments, the fixed bed is a structured fixed bed (whichmeans that it is formed of an easily replicated, generally homogeneous,substantially fixed structure, and thus is not randomly oriented orunstructured, and, as can be appreciated, could take a variety of sizesor shapes while meeting this qualification). In some embodiments, thestructured fixed bed described herein can provide for a large cellgrowth surface within a small volume while still allowing circulation ofmedium and cells. In some embodiments, the structured fixed beddescribed herein can comprise a tortuous path for cells and cell culturemedia. In some embodiments, a spacer layer facilitates the tortuouspath. In some embodiments, the structured fixed bed can comprise one ormore cell immobilization layers having a surface which allows cells toadhere and grow upon and forming a cell immobilization section. In someembodiments, adjacent to the cell immobilization layers are one or morespacer layers. In some embodiments, the spacer layer can include astructure which forms a spacer section. In some embodiments, the spacersection allows passage of cells and medium through an open but tortuouspath. In some embodiments, the structure or nature of the spacer layerscan be chosen such that the spacer layers create a tortuous, open pathfor cells and culture media to travel in parallel to the surface of saidspacer and cell immobilization layers. In some embodiments, the tortuouspath or channel formed by the spacer section creates turbulence whichfacilitates cell and cell medium incursion into the immobilizationlayers.

In some embodiments, the spacer layer can be a mesh or comprises a meshstructure. In some embodiments, mesh structure or mesh can be astructure comprising a network or web-like pattern of filament, wire orthread. In some embodiments, the network can define pores, openings orperforations formed of a three-dimensional weave. In some embodiments,the spacer layers and/or the cell immobilization layers of a spacersection and a immobilization section can be made of a biocompatiblepolymer, for example polyester, polyethylene, polypropylene, polyamide,plasma treated polyethylene, plasma treated polyester, plasma treatedpolypropylene or plasma treated polyamide. In some embodiments, thespacer layer or the cell immobilization layer can comprise silica,polystyrene, agarose, styrene divinylbenzene, polyacrylonitrile orlatex. In some embodiments, the layers can be hydrophilic orhydrophobic. In some embodiments, the cell immobilization layer can behydrophilic. In some embodiments, a cell immobilization layer can bewoven or nonwoven. In some embodiments, a cell immobilization sectionand a spacer section can be alternately positioned. In some embodiment,alternately positioned sections can alternate in a vertical position orin a horizontal position. In some embodiments, one or more layers ofcell immobilization layers can be superimposed on one or more spacerlayers (or vice versa). In some embodiments, a structured bed disclosedherein can be tightly or loosely rolled to a structure such as a spiralstructure or varying shape.

Reference is now made to FIGS. 1-3 , which illustrate one embodiment ofa bioreactor 100 for culturing cells, according to one aspect of thedisclosure. In some embodiments, the bioreactor 100 includes an externalcasing or housing 112 forming an interior compartment and a removablecover 114 for covering the interior compartment, which may includevarious openings or ports P with removable covers or caps C for allowingfor the selective introduction or removal of fluid, gas (including byway of a sparger), probes, sensors, samplers, or the like. As indicatedin FIGS. 2A, 2B, and 2C, in some embodiments, the bioreactor 100 may beused in connection with an external reservoir 102 and conduits 104(e.g., forward and return) to form a continuous loop for circulatingfluid to the bioreactor 100.

Within the interior compartment formed by the bioreactor housing 112,several compartments or chambers may be provided for transmitting a flowof fluid or gasses throughout the bioreactor 100. As indicated in FIG. 3, in some embodiments, the chambers may include a first chamber 116 ator near a base of the bioreactor 100. In some embodiments, the firstchamber 116 may include an agitator for causing fluid flow within thebioreactor 100. In some embodiment, the agitator may be in the form of a“drop-in” rotatable, non-contact magnetic impeller 118 (which asoutlined further below may be captured or contained within a container(not shown) including a plurality of openings for admitting andreleasing fluid). In some embodiments, as a result of the agitationprovided, fluid may then flow upwardly (as indicated by arrows A in FIG.2 ) into an annular chamber 120 along the outer or peripheral portion ofthe bioreactor 100. In some embodiments, the bioreactor is adapted toreceive a fixed bed, such as a structured spiral bed 122, which in usemay contain and retain cells being grown. As indicated in FIG. 3 , insome embodiments, the spiral bed 122 may be in the form of a cartridgethat may be dropped or placed into the chamber 120 at the point of use.In some embodiments, the spiral bed 122 can be pre-installed in thechamber during manufacture at a facility prior to shipping.

In some embodiments, fluid exiting the chamber 120 is passed to achamber 124 on one (upper) side of the bed 122, where the fluid isexposed to a gas (such as oxygen or nitrogen). In some embodiments,fluid may then flow radially inwardly to a central return chamber 126.In some embodiments, the central return chamber can be columnar innature and may be formed by an imperforate conduit or tube 128 or ratherformed by the central opening of the structured spiral bed. In someembodiments, the chamber 126 returns the fluid to the first chamber 116(return arrow R) for recirculation through the bioreactor 100, such thata continuous loop results (“bottom to top” in this version). In someembodiments, a sensor, for example a temperature probe or sensor T mayalso be provided for sensing the temperature of the fluid in the chamber126. In some embodiments, additional sensors (such as, for example, pH,oxygen, dissolved oxygen, temperature) may also be provided at alocation before the fluid enters (or re-enters) the chamber 116.

FIG. 3A shows one embodiment of a matrix material for use as astructured fixed bed in the bioreactor of the present disclosure and, inparticular, a spiral bed 122. In some embodiments, one or more cellimmobilization layers 122 a are provided adjacent to one or more spacerlayers 122 b made from a mesh structure. In some embodiments, thelayering may optionally be repeated several times to achieve a stackedor layered configuration. In some embodiments, the mesh structureincluded in spacer layers 122 b forms a tortuous path for cells (seecells L in FIG. 3B suspended or entrapped in the material of theimmobilization layer 122 a), and a cell culture may form part of anyinvention claimed herein) and fluid to flow when layered between twoimmobilization layers 122 a. Homogeneity of the cells is maintainedwithin the structured fixed bed as a result of this type of arrangement.In some embodiments, other spacer structures can be used which form suchtortuous paths. In some embodiments, as shown in FIG. 3A, the structuredfixed bed can be subsequently spirally or concentrically rolled along anaxis or core (e.g., conduit 128, which may be provided in multiplecomponent parts). In some embodiments, the layers of the structuredfixed bed are firmly wound. In some embodiments, the diameter of thecore, the length and/or amount of the layers will ultimately define thesize of the assembly or matrix. In some embodiments, thickness of eachof the layers 122 a, 122 b may be between 0.1 and 5 mm, 01 and 10 mm, or0.001 and 15 mm.

According to one aspect of this disclosure, the bioreactor 100 incertain embodiments may be “modular.” In some embodiments, a modularbioreactor can be comprised of a plurality of discrete modules thatinteract together to create a space suitable for culturing cells in amanner that is highly predictive due to the manufacturing homogeneity ofthe modules. In some embodiments, a modular bioreactor is not limited toparticular shape or form (e.g., cylindrical or otherwise, and with astructured fixed bed or unstructured bed, depending on the application).For example, as shown in FIG. 4 , In some embodiments, the modules maycomprise a base portion formed by base module 130, an intermediateportion formed by an intermediate module 140 (which may be formed from anumber of stackable modular portions, as outlined further in thedescription that follows), an optional associated central module, suchas conduit or tube 128, which may also be considered part of theintermediate module, and a cover module, such as formed by a cover partin the form of lid or removable cover 114. In some embodiments, themodules may be separately manufactured as individual components andeither assembled at a manufacturing facility based on an intendedapplication (and then shipped to a point of use) or assembled based onan intended application at the point of end use. In some embodiments,the modules of the bioreactor 100 interact to create a place for growingcells, such as in a high-density manner using a fixed bed, such as forexample a structured or unstructured fixed bed.

A further embodiment of a bioreactor 200 according to the disclosure isshown in FIGS. 5-9 . In some embodiments, the bioreactor (whethermodular or otherwise pre-assembled as a single unit) can comprise abase, an intermediate portion and a cover. In some embodiments, a baseportion can comprise a base part 230. In some embodiments, anintermediate portion can comprise intermediate parts 250 and/or 270. Insome embodiments, intermediate parts 250 and 270 are not identical. Insome embodiments, a cover portion can comprise a cover part 280.Referring to FIG. 6 , in some embodiments, base part 230 may include anexternal wall 232 and an internal wall 234, which may define a firstchamber 216 for receiving the agitator (not shown). In some embodiments,the internal wall 234 can include openings 234 a for allowing fluid flowto the second, radially outward chamber 220 bounded by the external orouter wall 232.

As can be seen in FIG. 6 , in some embodiments, the internal wall 234may include a plurality of connectors, such as grooves 236, for engagingcorresponding connectors, such as tongues 250 a, on the firstintermediate part 250, as shown in FIG. 8 . In some embodiments, theinternal wall 234 may be of lower/higher height than the external wall232. In some embodiments, the internal wall 234 may be of lower heightthan the external wall 232, as can be seen in FIG. 8 . With reference toFIG. 5 , in some embodiments, the first intermediate part 250 may be atleast partially recessed within the base part 230.

In some embodiments, the base part 230 may include a peripheralconnector, such as a groove 237. In some embodiments, the connector orgroove 237 can be adapted to receive a corresponding connector of asecond intermediate part 270, which may simply be part of an outer wall262 thereof. In some embodiments, within the intermediate part 270 canbe located a plurality of fixed beds 274 in a third chamber 224 (but asingle monolithic fixed bed could be used, which in this or anydisclosed embodiment may take any size, shape, or form), which could besupported by an interposed support, but a gap G could also be providedbetween adjacent sections of fixed beds). The gap could also beeliminated, such that an upper bed rests on and is supported by a lowerone.

In some embodiments, the structured fixed bed can be of the spiral form,as shown in FIGS. 3, 3A, 3B, and 3C (which spiral form can beimplemented in any embodiment of a bioreactor, disclosed or otherwise).In the case of a spiral bed, the bed may be wound around an internalwall 266, which may form a fifth chamber 228 for returning fluid to thefirst chamber 216 in the base part 230. The internal wall 266 maycomprise multiple stacked tubular parts, as shown. In some embodiments,the multiple stacked tubular parts can allow for the height to beadjusted depending on the number of fixed beds present (e.g., onetubular part may be provided for each stacked bed).

In some embodiments, the cover part 280, or lid can be adapted toremovably connect with the second intermediate part 270, and thus form afourth chamber 226 in which the liquid encounters gas, for example air.In some embodiments, the connection between the cover part and thesecond intermediate can be by a connector, such as a groove 282, whichreceives the upper end of the outer wall 262 or any access mechanismdisclosed herein. The lid or cover part 280 may include various ports P.

Turning back to FIGS. 7 and 8 , further details of the intermediate part250 are shown. In some embodiments, part 250 may include a plurality ofradially extending supports 254, which thus lend support for astructured fixed bed when resting thereon in the adjacent third chamber224. In some embodiments, supports 254 may also support a lower shelf256 defining a partial opening O for allowing fluid to flow vertically.In some embodiments, the height H of the supports 254 can be sufficientto allow the fluid to develop sufficient upward velocity before enteringthe chamber 224 to pass through the full section of the fixed bed 274.

In some embodiments, an inner annular wall 258 can be connected to theinboard end of the supports 254. In some embodiments, the wall 258,corresponds in diameter to the diameter of the internal wall 266 of theintermediate part 270, which may also connect with it (such as bynesting). In some embodiments, the internal wall 266 can form a passagefor delivering fluid from the fifth chamber 228 to the first chamber216. In some embodiments, a flow disruptor 260 may be provided in thispassage to help prevent the creation of any vortex within the fifthchamber 228.

In some embodiments, it may be desirable to provide one or more of theports P on the cover part 280 with internal threading in order toestablish a threaded connection with a component, such as a sensor (notshown). Thus, according to a further aspect of the disclosure, and withreference to FIGS. 9 and 10 , the cover part 280 may be formed byproviding a metal insert 292 with a helical thread into an injectionmold 294, and then injecting a plastic material into the mold to form acomposite part. In some embodiments, the threads may be reliablyprovided in the cover part 280, which may otherwise be formed ofplastic. As can be appreciated, this technique may also be used inconnection with any other parts of the disclosed bioreactors requiringthreaded fittings or ports. In some embodiments, inserts for use in thistechnique may be obtained from Wilhelm Böllhoff GmbH & Co. KG ofBielefeld, Germany, under the IMTEC brand.

From FIG. 5 , in some embodiments, it can be understood that the flowfrom one fixed bed module to the next-adjacent fixed bed module in thecell culturing chamber 224 can be direct or uninterrupted. In someembodiments, the outer chamber 224 can create a continuous flow paththrough the multiple beds located therein, which may be structured fixedbeds, unstructured fixed beds, or unstructured beds. In someembodiments, the continuous and substantially unimpeded flow through thepredesigned and matching bed modules helps to promote homogeneity forcell growth and other processing and enhances the consistency of thecell culturing operation, and also promotes the ability to takemeasurements or samples from the stacked beds, which is not readilypossible if blocking partitions (as contrasted with the perforatedsupports, as discussed below) are present. Finally, in a structured bedembodiment, the manufacture of the overall bioreactor is even lesscomplicated and labor intensive as the effort to match the propertiesand characteristics from one fixed bed module to the other is greatlyreduced.

Reference is now made to FIGS. 11 and 12 , which schematicallyillustrate a third embodiment of a bioreactor 300, which for purposes ofclarity is shown in cross-section. In some embodiments, the bioreactor300 (whether modular or otherwise pre-assembled as a single unit)comprises an external housing 331 with a cover 333, either of which mayinclude various openings or ports for allowing for fluid introduction orremoval. In some embodiments, within the bioreactor housing 331, severalcompartments or chambers are provided, including a first chamber 316including an agitator for causing fluid flow within the bioreactor 300,which may be in the form of a “drop-in” rotatable, non-contact magneticimpeller 318 or an agitator disclosed herein. As indicated in FIG. 11A,in some embodiments, the impeller 318 may be housed, captured orcontained within a housing, such as a housing or container 318 aincluding a plurality of openings 318 b serving as inlets and outletsfor admitting and releasing fluid (but any other form of agitator couldbe used). In some embodiments, the agitation created may be such thatfluid is caused to flow into a second or outboard annular chamber 320,which is radially outward of the first chamber 316.

In some embodiments, fluid may then flow upwardly (as indicated byarrows in FIG. 12 ) into a third annular chamber 324 along anintermediate, outer portion of the bioreactor 300. In some embodiments,the outer portion can be adapted to receive a fixed bed, such as astructured spiral bed 325, but other forms may be used), which in usemay contain cells being grown. In some embodiments, the spiral bed 325may be in the form of a cartridge that may simply be dropped into thechamber 324 at the point of use, or could be pre-installed in thechamber during manufacture at a facility prior to shipping.

In some embodiments, fluid exiting the third chamber 324 can then passedto a fourth chamber 326, where it is exposed to a gas (such as air) andthen flows radially inwardly to a fifth chamber 328, which is columnarin nature and returns the fluid to the first chamber 316 forrecirculation through the bioreactor 310, such that a continuous loopresults. In some embodiments, a temperature probe or sensor T, or anyother sensor disclosed herein may also be provided for sensing aparameter, for example the temperature of the fluid directly in thefifth chamber, and additional sensors (such as, for example, pH ordissolved oxygen) may also be provided at this location (which is beforethe fluid enters (or re-enters) the fixed bed 325).

From the partially cutaway image at FIG. 11B, it can be understood thatthe third chamber 324 may be bounded by upper and lower plates 330, 332,which include openings or perforations for allowing fluid generally freeof cells to enter and exit the fixed bed 325. In some embodiments, thelower plate 332 may include a central opening 332 a for allowing fluidto pass from the fifth chamber 328 to the first chamber 316 forrecirculation. In some embodiments, the upper plate 330 can include anopening 330 a, into which fluid may travel to enter the fifth or returnchamber 328.

In some embodiments, support for the upper plate 330 may be provided bya hollow, generally cylindrical tube 334, but could take other shapes.In some embodiments, the opposed ends of this tube 334 may fit intocorresponding grooves 330 b, 332 b in the plates 330, 332 (in some casesthe lower plate 332 can be integral with the impeller housing orcontainer 318 a in the illustrated embodiment). In some embodiments,supports, such as generally vertical rods 336, can be arranged toprovide added support for the plate 330. In some embodiments, thedisclosed vertical rods 336 do not interfere in any significant way withthe fluid flow in the corresponding chamber 328. In some embodiments,the ends of the rods 336 may be recessed in the plates 330, 332, or heldin place by suitable fasteners or locking mechanisms (e.g., lockingconnections, bolts or adhesives).

From FIG. 12 and the action arrows provided thereon, it can beunderstood that, as a result of the fluid agitation, in someembodiments, fluid may flow from the chamber 316 outwardly into chamber320. In some embodiments, the fluid can then be redirected to passvertically through chamber 324 including the fixed bed, and into chamber328. In some embodiments, fluid is then directed inwardly to chamber328, where the fluid may return to the first chamber 316 via opening 332a. In some embodiments, fluid can refer to culture medium.

FIG. 13 further illustrates an arrangement in which, in someembodiments, the upper plate 330 is provided with peripheral openings330 c to allow fluid to flow directly along the inner wall formed bytube 334. In this manner, a thin layer or film of fluid may be created,which flows downwardly while passing through the fifth chamber 328. Insome embodiments, this may serve to increase the volume of the fluidexposed to gas (air) within the fifth chamber 328, prior to it beingreturned to the first chamber 316. In some embodiments, thisimplementation can allow for more oxygen transfer which may be neededfor larger sizes or otherwise to increase cell growth rates adjustprocess parameters based on the biologic being produced.

In some embodiments, the “waterfall” implementation that creates a fluidfilm can be achieved by adding a limited quantity of cell culture mediumfrom the start, such that only a small overflow results. Alternatively,in some embodiments, the “waterfall” implementation is achieved byadding cell culture medium and cells and then when cells are growing inthe bed, withdraw culture medium (such as using a dip tube) in thecorresponding chamber, such as chamber 328.

In some embodiments, a fourth embodiment of a bioreactor 400 isdescribed with reference to FIGS. 14-16 . In this embodiment, thebioreactor 400 includes the first through fifth chambers 416, 420, 424,426, and 428 as noted above (fixed bed not shown), but the housing 412is comprised of a plurality of modular parts. In some embodiments, theparts include a base part 430, one or more intermediate parts 450, and acover part 470. In some embodiments, the parts 430, 450, 470 can beadapted to interact in a fluid-tight manner so as to form the bioreactor400 with the chambers 416, 420, 424, 426, and 428, as noted.

In some embodiments, and as perhaps best understood from FIG. 14 , thebase part 430 can include a peripheral connector, shown in the form of agroove 432, for receiving and engaging a corresponding peripheralconnector, such as a tongue 452, projecting from one of the intermediateparts 450. In some embodiments, interiorly, the base part 430 caninclude an upstanding wall 434, which defines the first chamber 416 forreceiving a fluid agitator (not shown). In some embodiments, the wall434 can includes openings or passages to allow for fluid to flowradially into an outer portion of the base part 430, which defines afurther or second chamber 420. In some embodiments, as the flow isredirected vertically as a result of the presence of the base part 430,turbulence is created, which thus promotes mixing and homogeneity of thefluid throughout the bioreactor and thus enhances the cell culturingprocess.

Two intermediate parts 450 a, 450 b are shown as being stacked, with aperipheral connector (groove 454) of the first (lower) part 450 aengaging a corresponding connector (tongue 452) of the second (upper)part 450 b. As can be appreciated from FIG. 14 , in some embodiments,each intermediate part 450 a, 450 b can include an outer side wall 456supporting the tongue 452 and groove 454, respectively. In someembodiments, radially inwardly, an inner wall 458 carries inner andouter connectors, which may be in the form of upstanding ledges 460,462, can be provided for receiving the corresponding ends of a tube 436,which thus forms periphery of the fifth or return chamber 428.

In some embodiments, the first or lower intermediate part 450 a may alsoinclude openings, such as elongated arcuate slots 464, which at leastpartially receive connectors, of the base part 430, such as upstandingprojections 434 a from the wall 434. In some embodiments, an interiorledge 466 can form central openings 466 a in the intermediate parts 450a, 450 b for permitting fluid to flow in an inner column defined by thewall 434, as well as to receive any temperature sensor, dip tube or thelike (which would be positioned after the fluid exits the fixed bed). Insome embodiments, the second intermediate part 450 b may be similarlyconstructed to promote interchangeability, in which case the openings(slots 464) in the second or upper intermediate part 450 b allow for thecreation of the thin falling flow or film of fluid within the fifth orreturn chamber 428, as previously noted.

In some embodiments, extending between the inner and outer walls 456,458 are a plurality of supports 468. In some embodiments, the supports468 include radially extending supports 468 a and at least onecircumferentially extending support 468 b, which together can create aperforated or reticulated plate-like structure that allows fluid flow(which structure in this or any embodiment may comprise a screen, net,grid, or other skeletal structure, and may be rigid, semi-rigid, orflexible). In fact, the supports 468 may be designed to enhance fluidflow through the bed(s) by maximizing the amount of open space createdby the openings for permitting fluid to pass. In some embodiments, forculturing cells, a fixed bed, such as the spiral bed (not shown) woundaround wall 434 may be positioned in the chamber 424 formed between theparts 450 a, 450 b. In some embodiments, fluid passing from the upperintermediate part 450 b can enters the fourth chamber 426 definedpartially by cover part 470, and may flow to the column forming thefifth chamber 428 before returning to the first chamber 416 forrecirculation.

In some embodiments, the cover part 470 includes a connector, such astongue 472, for fitting into the corresponding connector (groove 454) ofthe second intermediate part 450 b. In some embodiments, the cover part470 can also include a first or central receiver, such as upstandingwall 474 for receiving a removable cap or lid 476, which may includevarious ports P for connecting with conduits for delivering fluids orother substances to the bioreactor 400 (and the fifth chamber 428). Insome embodiments, the cap or lid 476 may also carry the temperaturesensor or probe T, as shown, as well as other sensors, and may also beadapted for providing additions or removing substances from thebioreactor 400, or for regulating a product manufacturing process. Ascan be appreciated, in some embodiments, the cap or lid 476 can be wellpositioned to allow for sensing or fluid sampling to occur in connectionwith the return flow via chamber 428. In some embodiments, a secondperipherally positioned receiver, such as upstanding wall 477, may alsobe adapted for connecting with a second cap or lid 478 for receivingsensors or depositing or withdrawing substances (including culturesamples) from the bioreactor and, in particular, a peripheral portionthereof including the third chamber 426 in which cell culturing iscompleted. In some embodiments, the caps or lids 476, 478 may havedifferent types of ports P and may be different sizes/shapes, or theymay be identical to promote interchangeability.

By comparing FIG. 14 with FIG. 5 , it can also be appreciated that thecap or lid 476, 478 may be used in connection with different sizes ofbioreactors. Thus, in FIG. 14 , it can be understood that the cap or lid476, 478 has an outer diameter that is much less than an outer diameterof the bioreactor 400. In some embodiments, cap or lid 476, 478 couldalso be used with the bioreactor 300 of FIG. 5 (or any other), in whichcase the outer diameter would be about the same or perhaps even slightlygreater than the diameter of the bioreactor 300.

In some embodiments, adhesives or glue may be used at the connections tohold the structures together. In some embodiments, threaded or locking(e.g., bayonet style) connections may also be used, such that afluid-tight seal is maintained to prevent leakage and help ensure thatsterility is maintained. In some embodiments, the arrangement of modularparts 430, 450, 470 allows for the bioreactor 400 to be pre-assembled,assembled or constructed on site rapidly, and potentially disassembledwith similar rapidity. As it is possible to easily add additionaltube(s) to form a heightened wall 434 or intermediate parts 450, thenumber of fixed beds or height of the bioreactor 400 may be adjusted tosuit a particular need or process setting depending on the application.

In some embodiments, the flow from one fixed bed to the next-adjacentone in the chamber is direct or uninterrupted. In some embodiments, theouter chamber 424 for receiving the bed creates a continuous flow paththrough the multiple beds present therein, which may be structured fixedbeds, unstructured fixed beds, or other beds. In some embodiments, thecontinuous and substantially unimpeded flow helps to promote homogeneityas if the modules are actually a single bed and thus improves thepredictability and quality of the cell culturing process. Homogeneitymeans that the cell distribution throughout the bed is homogeneous orhaving a somewhat equal spread.

FIG. 16 illustrates an alternative embodiment of an intermediate part450, which can be adapted for positioning above the base part 430. Insome embodiments, a plurality of radially extending supports 466 b areprovided in the central opening 466 a, which connect with an interiorconnector in the form of a ring 466 d. In some embodiments, the ring 466d may be sized to receive part of a carrier 480 for carrying theagitator (not shown), and thus suspending it above the floor of the basepart 430. In some embodiments, based on the structure, friction andconcomitant particle shedding as a result of frictional contact betweenthe impeller and the floor of the base part 430 during rotation isavoided.

As illustrated, in some embodiments, the carrier 480 may comprise a pairof compressible clips 482, which may be squeezed together to passthrough opening in the ring 466 d, and then released to securely suspendthe carrier from the intermediate part 450, while permitting relativemovement that allows the carrier to rotate freely. In some embodiments,the carrier 480 may include a socket 484, shown as being C-shaped incross section, that receives a corresponding portion of the agitator,such as impeller (not shown) or perhaps simply an elongated magnetic orferromagnetic rod (not shown). In some embodiments, this portion maycomprise an upwardly extending projection rotatably connected to theagitator by a bearing. As can be appreciated, in some embodiments, thesocket 484 can allow for side-to-side movement of the agitator, as maybe necessary to achieve alignment with a corresponding external ornon-contact (e.g., magnetic) drive D located external to the bioreactor400, such as below the base part 430.

FIGS. 16A and 16B also illustrate an alternate embodiment of a modularbioreactor 400 including fixed beds 496. In some embodiments, the basepart 430 and cover part 470 can be adapted for connecting with an outercasing 492, which creates a gap or space with the periphery of theintermediate parts 450. In some embodiments, the gap G or space may beused for providing a heating or cooling effect to control thetemperature of the beds associated with the intermediate parts 450. Thegap G or space may also simply supply insulation of the walls of theintermediate area of the bioreactor which are close to growing cellswithin the bed and likely to be sensitive to temperature variations.This insulation acts to prevent heat which is applied to the bottom ofthe base part 430 of the bioreactor from extending up to the adheredcells in the bed(s) 496.

FIG. 16A also illustrates the possible use of sparging in thebioreactor, which may be provided in any disclosed embodiment. In theillustrated arrangement, the sparging is provided by a sparger 494located in the fifth chamber 428. The bubbles generated as a result maythus flow upwardly countercurrent to the return fluid flow.

These figures, and perhaps FIG. 16B best, also show that theintermediate parts 450 may engage internal tubes 436, which are fluidimpervious to thus provide the chamber 428 for returning flow to thebase part 430, where it may be agitated and returned to enter the bedsfrom below and flow upwardly therethrough (in any embodiment disclosed).These tubes 436 may be provided such that one tube corresponds to eachfixed bed 496 present, as shown, and two intermediate parts 450 engageeach tube 436 (e.g., one from below and one from above). However, inthis or any other disclosed embodiment, it should be appreciated thatthe innermost surface of the fixed bed, such as the innermost spiralwrap of a spiral bed, may be made to perform a similar function bymaking it or otherwise conditioning it so as to be impervious to fluid.For instance, the surface may be coated with a fluid-impervious orhydrophobic material, such that it still retains the fluid in the bed(s)and maintains a distinct, return flow of fluid through the centralcolumn formed by chamber 428.

FIG. 16C also illustrates an embodiment of the bioreactor 400 includingthe intermediate parts 450 a, 450 b sandwiching a fixed bed 496, whichmay be a structured, spiral bed as previously illustrated and described.The base part 430 and cover part 470 are also provided and interfacewith the outer casing 492, creating an annulus or gap, which again maybe insulated or associated with a heating or cooling means. In this orany other embodiment, the casing 492 may simply create a buffer or space(filled with air or other gas). This may allow for the temperature ofthe bioreactor 400 to be regulated more efficiently (e.g., quicker) andfurther allows it to be perfused and/or used in media recirculation witha lower requirement in term of media pre-heating.

This figure also illustrates the housing 418 for an agitator 418 a. Thehousing 418 may be any one of the forms shown in FIG. 21 , and thus mayinclude a flow divider 418 d. The inner partition in the form of tube436 for partially forming the central column (i.e., return chamber 428shown in FIG. 16A) is also shown. An outer partition is also shown, mayalso be in the nature of a cylindrical structure or tube 496 thatremovably interconnects with the parts 450 a, 450 b (and may be adheredin place using adhesives or other forms of bonding), but could also be aunitary structure with one or both of them.

FIGS. 17 and 18 illustrate an example of a bioreactor 500 including oneor more fixed beds, such as two vertically stacked, structured fixedbeds 518 a, 518 b in the illustrated example. In some embodiments, thebeds 518 a, 518 b can be arranged in an outer chamber 512 a of thebioreactor 500 and may be the spiral beds shown in FIGS. 1-3 . In someembodiments, an inner chamber 512 b can also provide circulating fluidto or from the fixed bed(s). In some embodiments, the fluid may becaused to flow by an associated agitator, such as an impeller 520located in a lower compartment 512 c of the bioreactor 500. In someembodiments, the flow of fluids may be in a vertical direction withinthe fixed bed(s), such as from top to bottom or bottom to top. In someembodiments, the structured fixed bed(s) can be provided in the innerchamber 512 b, with the outer chamber 512 a serving to deliver fluid toand from the inner chamber.

Referring now to FIGS. 19 and 20 , in some embodiments, an agitator inthe form of an impeller 600 can be used in any of the above describedembodiments is shown. In some embodiments, the impeller 600 may comprisemagnets 602 that can be inserted into a body 604 (machined or injected)having radially extending blades 606, and an opening 608, and throughwhich a shaft 610 or other receiver can be inserted. In someembodiments, caps (not shown) may be provided over the magnets 602 toensure that contact is not made with the culture media, and may beattached using an adhesive or threaded connection. In some embodiments,the magnets 602 can be overmoulded when the body 604 embodiment will beinjected (injection molded). In some embodiments, it may also bepossible to 3D print the embodiment, to pause the 3D printing, to insertthe magnets, and to relaunch the 3D printing to form the impeller 600.In some embodiments, the impeller body 604 may be made in a durable,polymer material, such as polycarbonate or other suitable materials. Insome embodiments, the impeller can be machined, injection molded, 3Dprinted, or fabricated in or other ways. The associated receiver orshaft 610 (if present) may be formed of polypropylene or other suitablematerials, and may be machined, injected or 3D printed.

FIG. 21 shows various combinations of impellers 600 with differentcontainers 618 a in a table form, with an indication of the relativeefficiencies that result. In some embodiments, by adjusting the radialextent of the blades B and changing the number of outlets O in thecontainer 618 a to more than four (and possibly as many as 10-12), ahigher efficiency in terms of fluid flow may be realized at a comparablerotational speed. In some embodiments, a divider, such as an upstandingwall 618 d having an X-shaped cross-section may be provided adjacent tothe inlet I of the container 618 a for dividing the flow. In the twoembodiments at the right of FIG. 19 , it can also be understood thatvanes V are providing for guiding the flow as it exits the container 618a and, as indicated, the vanes can have varying shapes or widths.

FIG. 22 further illustrates a further example of an impeller 650 havingblades B that curve in a radial direction. In some embodiments, theimpeller 650 may include a central space 651 for receiving flow from theinlet I of the container 618 a when used in connection with such, andthe blades B thus serve to redirect the fluid outwardly through theoutlets O. The impeller 650 is shown as having 10 blades, but more orfewer may be provided as desired or necessary. In some embodiments, theimpeller 650 may also include one or more magnets (not shown), asdescribed above, for forming a non-contact coupling with an externaldrive (not shown). Because living cells are sensitive to mechanicalforces such as shear, the impeller design needs to avoid shear whileproviding for efficient and optimized fluid flow. The impeller 650achieves such complimentary goals.

Any of the components of the above bioreactors 100-500 may be made to bea single use or disposable component, or may be made to be reusable.Furthermore, the components used may be a mix or hybrid of disposableand reusable materials. In some embodiments, the bioreactor 100-500 mayhave a diameter of approximately 50-60 cm. In some embodiments, thebioreactor 100-500 may have a diameter or height of approximately morethan about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90 or 100cm. In some embodiments, the cover part or lid 476, 478 that may be usedin connection with bioreactor 400 may have a diameter of approximatelymore than 2, 4, 5, 6, 8, 10, 12, 15, 20, 25, 30 or 50 centimeters. Insome embodiments, the intermediate parts 450 a, 450 b may have a heightof approximately about 2.5-5.0 centimeters or more. In some embodiments,the overall bioreactor 400 may have a height of approximately 20-50centimeters. In some embodiments, a bioreactor can comprise more thanone fixed bed. In some embodiments, an impeller speed may be adjusted tocompensate for an increase in pressure drop so as to maintain consistentlinear velocity from bottom of reactor to top of reactor. In such case,shear stress on cells can be maintained constant for all sizes ofbioreactor. In some embodiments, a sparger may also be provided. In someembodiments, it may be desirable during sparging to cease operation ofthe impeller to avoid transporting the air bubbles into the fixed bed.

In some embodiments, in the modular case, the bioreactor 100, 200, 300,400, 500 may comprise any number of components for adjusting therelative height thereof. For example, a plurality of intermediate parts,such as parts 450, may be used to create an increased height. In someembodiments, the bioreactors 100, 200, 300, 400, 500 may also beprovided in a number of different diameters, and each diameter maycomprise one or more intermediate parts for creating different heightsbased on a particular application. In some embodiments, the fixed bedgrowth surfaces may range from <1 m² to 2 m², 7-30 m², 150-600m², >2,400 m², and may vary among different sizes (height or diameter)of bioreactors. As noted, a plurality of fixed beds may be provided in astacked configuration, such as one, two, three, four, or more fixedbeds.

In some embodiments, in the above-described “waterfall” arrangements, itmay be desirable to increase the oxygen transfer (or kLa, the volumetricmass-transfer coefficient that describes the efficiency with whichoxygen can be delivered to a bioreactor for a given set of operatingconditions) by providing a degree of turbulence as the fluid passes intothe inner or central column. To achieve this result, one or more flowdisruptors may be provided to interrupt the laminar flow and cause it tobecome turbulent. FIGS. 23 and 24 illustrate a further possiblemodification for the modular bioreactor, in which the flow disrupters ordividers may be provided as upstanding projections 702 on a ring 700(thus forming a crown) which may be located above the central column.Consequently, fluid flow otherwise entering the central column 736 as afilm may be “broken” by the projections 702, which thus form individualstreams that are more turbulent and enable better oxygen transfer. Insome embodiments, the projections 702 can break the potential swirlingmovement upon leaving the fixed bed, and ensure that the fluid flow canbe aligned with the center of the bioreactor.

Turning to FIGS. 25 and 26 , it can be understood that the resultingindividual flows may ultimately recombine within the central column orcolumnar region formed by the inner wall of structured fixed bed, whichmay lead to added turbulence. Furthermore, it can be further understoodthat the ring 700 may cause the flow to assume a parabolic trajectoryinto the column, which can create a pocket P below the flow, whereair/oxygen may become trapped. In some embodiments, to allow for gaseousexchange to occur between this pocket P and the interior of thebioreactor above the central column, one or more conduits 704 may beprovided. In FIG. 25 , a single conduit 704 is shown, which thus formsan inlet for gas flow. As shown in FIG. 25 , multiple conduits 704 a,704 b may be provided, and may serve as inlets and/or outlets for gas,such that it is renewed. As further indicated, the conduits 704 may beintegral with the ring 700, as shown in FIG. 25 , or may be separatefrom it, as shown in FIG. 26 .

Turning now to FIGS. 27 and 28 , a disposable (e.g., plastic or polymer)connector 800 for connecting a non-disposable (e.g., stainless steel)probe 802 for sensing various conditions of the bioreactor 100, 200,300, 400, 500 is shown. In some embodiments, the connector 800 maycomprise a tube or sleeve 804 associated with a cap or cover 806 at oneend, and an adaptor 808 at the other, which may be for connecting with aport in any wall or portion of the bioreactor 100, 200, 300, 400, 500such as by way of a threaded connection. In some embodiments, anoptically transmissive portion, such as a membrane 810 attached to thecap 806, may be provided for interfacing with the probe 802.

EXAMPLES

The following examples are given for the purpose of illustrating variousembodiments of the invention and are not meant to limit the presentinvention in any fashion. The present examples, along with the methodsdescribed herein are presently representative of exemplary embodiments,and are not intended as limitations on the scope of the invention.Changes therein and other uses which are encompassed within the spiritof the invention as defined by the scope of the claims will occur tothose skilled in the art. The following example is provided toillustrate the efficacy of the disclosed bioreactor.

A bioreactor was prepared per FIG. 1 by hydrating a structured fixed bedunder aerated and agitated conditions (750 mL culture medium ensuring afalling film of ˜5 cm, 740 rpm (corresponding to a vertical linearvelocity of 1 cm s⁻¹ through the fixed bed), 1 hour hydration time).Medium equilibration was achieved by setting the temperature and pH atthe experimental set points (respectively 37° C., 7.2) and starting theautomated regulations. DO calibration was first achieved undernon-regulated, aerated conditions (100% DO set point), subsequently towhich the DO regulations (>50%) was started. Probe calibration (on andoffline measurements) was performed once the medium had reached the setoperating temperature. Inoculum was manually transferred to thebioreactor at an initial seeding density of 3.0×10⁴ cells cm⁻² underagitated conditions, ensuring that the total liquid volume remainedconstant by removing the corresponding volume of equilibration medium.Culture parameters recording (pH, DO, T) was started immediatelythereafter. An external media source was connected and recirculatedthrough the bioreactor shortly after inoculation, per FIG. 2 .

Using this experimental set up, two cell cultures were performed for 3days in batch mode (infection cell density=1.9±0.2×10⁵ cells cm⁻²). Athird cell culture was performed for 6 days (infection celldensity=6.5×10⁵ cells cm⁻²) with the replacement at day 3 of theexternal media source with fresh material to ensure nutrientavailability. Infection was performed using an adenovirus (stock:1.7×10⁹ ifu mL⁻¹) at a set target cell density of infection and fixedMOI of 5. Harvest of the cell culture medium was performed using anoptimized detergent addition (Triton™ X-100; Sigma Aldrich, Overijse,Belgium) for in-situ bulk cell lysis under agitated conditions. Thefollowing summary is provided:

-   -   Bioreactor vessel effective working volume: 750 mL    -   Recirculation volume: 4.2 L    -   Agitation speed: 740 rpm (1 cm s−1 vertical velocity)    -   Falling film height: ˜5 cm    -   Innoculation density: 25,000-30,000 cells cm-2    -   pH: 72    -   Dissolved oxygen: 50%    -   Harvest treatment: Triton X-100, Benzonase and MgCl2−    -   Culture time: 3-6 days        Control adherent cultures in a CELLSTACK bioreactor were        performed under identical operating conditions where relevant        (temperature, media composition, initial pH, headspace gas        composition). FIG. 29 shows the cell growth curves in inventive        fixed bed bioreactor compared to their controls carried out in        CELLSTACK bioreactor. The cell density reached in the structured        fixed bed bioreactor is higher under the same conditions than in        the control experiment, both at day 3 and even more so at day 6        (6.5×105 vs. 3.2×105 cells cm−2). In order to achieve the high        10 cell density observed at day 6, 85% of the medium was        exchanged at day 3 in the CELLSTACK bioreactor, while an        external bottle containing 4.2 L of fresh culture medium was        connected and circulated (10 mL min−1) through the bioreactor,        corresponding to the same ratio of medium exchange. Infection        was performed at a target cell density in the cell culture. All        cells were observed to be lysed 3 days post infection, an        observation upon which it was decided to recover the product        using the detergent treatment. The same harvest protocol was        performed on the control with the CELLSTACK bioreactor and the        data is presented alongside the bioreactor runs in FIG. 30 .        Bioreactor #2 according to the disclosure was operated with an        external medium circulation loop of 4.2 L from the onset and        infected at day 3 (see Methods for further details—cell density        post-infection not shown). Bioreactor #1 according to the        disclosure was started just like bioreactor #2 except that at        day 3 the external media circulation loop was replaced with        fresh medium thus allowing further cell growth. Bioreactor #2        was infected at day 2 whereas bioreactor #3 was infected at        day 3. The error bar represents the range of the TCID50        analytical assay measurement. The productivity between the        inventive bioreactors and their control is similar and the        differences are within the error range of the assay.

A further experiment was conducted to demonstrate the efficacy of astructured fixed bed in a bioreactor, as shown in FIGS. 31 and 32 . Thecell culture conditions were as follows:

-   -   Vero cells    -   Media 5% serum-volume/surface ratio: 0.17 ml/cm²    -   Inoculation 5000c/cm²    -   5 days of growth    -   37° C.-pH: 7.2-DO=50%    -   Agitation of the bioreactor: 550 rpm    -   Recirculated batch mode with a flow of 20 ml/min

As can be appreciated, these figures illustrate that uniform cell growthwas achieved, both axially and radially. with the bioreactor using astructured fixed bed, and in particular, a spiral or “snail” bed.

Referring now to FIGS. 33-37 , a method of manufacturing a modularbioreactor 1000 having a structured fixed bed is disclosed. Turningfirst to FIG. 36 , the method may comprise associating a tubular part1002 with the structured fixed bed, which may be the spiral bed 1004previously described. The spiral bed 1004 may be formed by wrapping orwinding the matrix material around the tubular part 1002, which may befastened to a base 1006, such as by using a tongue and groovearrangement, and adhered in place. This forms a first stage bioreactorprecursor 1007.

Next, an upper portion 1008 may be secured to the base 1006, also usingan adhesive. This is done to arrive at a second stage bioreactorprecursor 1010, as shown in FIG. 38 , which further shows the use ofweights 1012 for ensuring proper seating of the parts and a UV lamp 1014for curing the adhesive.

With reference to FIG. 39 , a lid 1016 may then be applied to thebioreactor precursor 1010, and any probe or sampler 1018 installed. Thelid 1016 may interface with an outer housing 1020, which may beadhesively bonded together. Weights 1012 and the UV lamp 1014 may againbe used to form a secure connection.

With reference to FIG. 41 , it can be understood that prior toinstalling the lid 1016, a stacked configuration may be achieved byinstalling multiple first stage bioreactor precursors 1007 a, 1007 bonto a second stage bioreactor precursor 1010. The upper portion 1008 ofthe second stage bioreactor precursor 1010 may be adapted to receive aportion of the next adjacent first stage bioreactor precursor 1007 a,such as a depending portion of the tubular part 1002. Likewise, theupper portion 1008 of the first stage bioreactor precursor 1007 a may beadapted to interface similarly with the next adjacent second stagebioreactor precursor 1007 b. Lid and any probes, samplers, or the likemay be installed as noted above, and the bioreactor 1000 provided withmultiple stacked beds in this manner.

A further method of manufacturing a bioreactor is also contemplated. Inthis method, structured fixed beds are placed into an interiorcompartment or chamber of the bioreactor. As noted previously in thedisclosure, the beds may be in contact, or separated by a spacer (e.g.,a screen or like structure) or a gap.

In any embodiment, it can be appreciated that the monolithic nature ofthe structured fixed beds in the bioreactors 100, 200, 300, 400, 500help to promote consistency of the cell culturing operation throughout,and thus homogeneity. The ability to simply “drop in” a structured fixedbed into a bioreactor makes the cell culturing process easilyrepeatable, and the results subject to high reproducibility. This is asignificant advantage over known bioreactors using suspended orfluidized beds. As a result of the disclosed arrangements, cell culturehomogeneity (e.g., characteristics including temperature, pH and otherreagent characteristics) is maintained throughout. Homogeneity isfurther improved due to the impeller mixing and fluid flow.Homogenization is further improved due to the gaps wherere-homogenization (remixing) of the cell culture medium occurs. Even ifcell culture travels through the bed and it loses homogeneity, thathomogeneity is somewhat restored as the culture passes through the gaps.

Summarizing, this disclosure may also be considered to pertain to thefollowing items:

-   -   1. An apparatus for culturing cells in connection with a fluid        flow, comprising: a modular bioreactor including a fixed bed for        culturing cells.    -   2. The apparatus of item 1, wherein the modular bioreactor        comprises: a base portion having a first chamber,        -   an intermediate portion forming at least part of a second,            outer chamber for receiving the fixed bed and at least part            of a third inner chamber for returning fluid flow from the            second outer chamber to the first chamber, and        -   a cover portion for positioning over the intermediate            portion.    -   3. The apparatus of item 1 or 2, wherein the fixed bed comprises        a structured fixed bed.    -   4. The apparatus of any of the foregoing items, wherein the        intermediate portion comprises a tubular part, the structured        fixed bed extending spirally around the tubular part.    -   5. The apparatus of any of the foregoing items, wherein the        intermediate portion comprises a tubular part formed by an inner        wall of the fixed bed.    -   6. The apparatus of any of the foregoing items, wherein the        intermediate portion comprises a plurality of intermediate        parts, each associated with a structured fixed bed.    -   7. The apparatus of any of the foregoing items, wherein at least        one of the plurality of intermediate parts is perforated for        allowing fluid to flow from a first structured fixed bed below        the at least one intermediate part to a second structured fixed        bed above the at least one intermediate part.    -   8. The apparatus of any of the foregoing items, wherein each of        the plurality of intermediate parts is tubular, and each        structured fixed bed comprises a spiral bed wound around the        tubular intermediate part.    -   9. The apparatus of any of the foregoing items, further        including a perforated support for the structured fixed bed.    -   10. The apparatus of any of the foregoing items, wherein the        intermediate portion further includes a tubular casing for        forming a periphery of the modular bioreactor, the tubular        casing forming a space for heating, cooling, or insulating the        bioreactor.    -   11. The apparatus of any of the foregoing items, wherein the        intermediate portion comprises a plurality of intermediate        parts, each adapted for connecting with each other.    -   12. The apparatus of any of the foregoing items, wherein the        intermediate portion includes a tube for engaging at least one        intermediate part and forming an inner wall of the outer second        chamber for receiving the fixed bed.    -   13. The apparatus of any of the foregoing items, wherein the        tube engages a first intermediate part below the tube and a        second intermediate part above the tube.    -   14. The apparatus of any of the foregoing items, wherein the        second intermediate part includes openings for creating a fluid        film along the third inner chamber.    -   15. The apparatus of any of the foregoing items, further        including supports for supporting the second intermediate part        from the first intermediate part.    -   16. The apparatus of any of the foregoing items, wherein the        supports comprise vertical rods.    -   17. The apparatus of any of the foregoing items, wherein the        cover portion comprises a removable cap including a plurality of        ports.    -   18. The apparatus of any of the foregoing items, wherein the        removable cap has an outer diameter that is less than an outer        diameter of the intermediate portion.    -   19. The apparatus of any of the foregoing items, wherein at        least one of the ports includes a threaded metal insert.    -   20. The apparatus of any of the foregoing items, wherein the        cover portion has an outer diameter that is equal to or greater        than an outer diameter of the intermediate portion.    -   21. The apparatus of any of the foregoing items, wherein the        intermediate portion comprises an intermediate part adapted for        positioning at least partially within the base portion, the        intermediate part further including a flow disruptor for        disrupting fluid flow.    -   22. The apparatus of any of the foregoing items, wherein the        base portion includes a further chamber radially outward of the        first chamber in fluid communication with the second outer        chamber including the fixed bed, which is formed by an        upstanding wall having a plurality of openings for transmitting        fluid from the first chamber to the further chamber.    -   23. The apparatus of any of the foregoing items, further        including an agitator associated with the base portion.    -   24. The apparatus of any of the foregoing items, wherein the        intermediate portion is adapted for suspending the agitator in        the first chamber in a manner that allows side-to-side movement        for alignment with an external drive.    -   25. The apparatus of any of the foregoing items, further        including a container for containing the agitator, the container        including a central inlet and a plurality of radially oricntcd        outlets.    -   26. The apparatus of any of the foregoing items, wherein a flow        divider is associated with the central inlet.    -   27. The apparatus of any of the foregoing items, wherein the        agitator comprises a plurality of curved blades.    -   28. The apparatus of any of the foregoing items, further        including a plurality of flow disruptors for dividing the fluid        flow entering the third inner chamber into a plurality of        streams.    -   29. The apparatus of any of the foregoing items, wherein the        plurality of flow disruptors are associated with a ring.    -   30. The apparatus of any of the foregoing items, further        including one or more conduits for permitting gas to enter into        a space behind one of the streams.    -   31. The apparatus of any of the foregoing items, wherein the one        or more conduits are connected to a structure including the        plurality of flow disruptors.    -   32. The apparatus of any of the foregoing items, wherein a first        conduit is connected to the structure.    -   33. The apparatus of any of the foregoing items, wherein first        and second conduits are connected to the structure.    -   34. The apparatus of any of the foregoing items, wherein first        and second conduits are not connected to the structure.    -   35. An apparatus for culturing cells, comprising: a modular        bioreactor comprising a base portion connected to both a central        column and an outer casing, the outer casing and central column        together forming a compartment for culturing cells.    -   36. The apparatus of item 35, wherein the compartment includes        at least one structured fixed bed.    -   37. The apparatus of item 35 or item 36, wherein the compartment        includes a plurality of structured fixed beds, arranged in a        stacked configuration.    -   38. The apparatus of any of items 35-37, further including an        intermediate part between at least two of the plurality of        structured fixed beds.    -   39. The apparatus of any of items 35-38, wherein the at least        one structured fixed bed comprises a spiral bed.    -   40. The apparatus of any of items 35-39, wherein each of the        plurality of stacked, structured fixed beds is wrapped around        the central column.    -   41. The apparatus ofany of items 35-40, wherein the central        column comprises first and second interconnected tubes, a first        structured fixed bed of the plurality of structured fixed beds        being wrapped around the first tube and a second structured        fixed bed of the plurality of structured fixed beds being        wrapped around the second tube.    -   42. The apparatus ofany of items 35-41, wherein the central        column comprises first and second tubes for engaging a        perforated support extending between at least two of the        plurality of structured fixed beds.    -   43. The apparatus of any of the foregoing items, wherein the        fixed bed comprises a cartridge adapted for being inserted into        and removed from the second, outer chamber or compartment.    -   44. The apparatus of any of items 35-43, wherein the base        portion is removably connected to the central column.    -   45. The apparatus of any of items 35-, wherein the base portion        is removably connected to the outer casing.    -   46. A bioreactor for culturing cells, comprising:        -   a base part having a first chamber including an agitator for            agitating a fluid; and        -   a first central column removably attached to the base part,            the first central column forming at least part of a second,            outer chamber for culturing cells and a third inner chamber            for returning fluid flow from the second outer chamber to            the first chamber.    -   47. The bioreactor of item 46, wherein the second, outer chamber        includes a first structured fixed bed.    -   48. The bioreactor of item 47, wherein the first structured        fixed bed comprises a spiral bed.    -   49. The bioreactor of item 48, wherein the first structured        fixed bed is wound around the first central column.    -   50. The bioreactor of any of items 47-49, further including a        second central column forming at least part of the second outer        chamber, and further including a second structured fixed bed        spaced vertically from the first structured fixed bed.    -   51. The bioreactor of item 50, further including a perforated        support between the first structured fixed bed and the second        structured fixed bed.    -   52. The bioreactor of item 46, wherein the second, outer chamber        includes an unstructured bed.    -   53. A bioreactor for culturing cells in connection with a fluid,        comprising:        -   a first chamber including an agitator for agitating the            fluid;        -   a second, outer chamber including a plurality of stacked            beds for culturing cells; and        -   a third, inner chamber for returning fluid from the second            outer chamber to the first chamber.    -   54. The bioreactor of item 53, comprising:        -   a base portion having the first chamber;        -   an intermediate portion forming at least part of the second,            outer chamber and at least part of the third inner chamber;            and        -   a cover portion for positioning over the intermediate            portion.    -   55. The bioreactor of item 54, wherein the intermediate portion        comprises a first support for supporting a first bed of the        plurality of stacked beds.    -   56. The bioreactor of item 54 or item 55, wherein the        intermediate portion comprises a second support for supporting a        second bed of the plurality of stacked beds.    -   57. The bioreactor of any of items 54-56, wherein the        intermediate portion is adapted for removably connecting with        the base portion and the cover portion.    -   58. The bioreactor of any of items 54-57, wherein the second,        outer chamber is bounded by an outer wall, and further including        an outer casing forming a space with the outer wall, the space        being for insulating, heating, or cooling the second, outer        chamber.    -   59. A bioreactor for culturing cells in connection with a fluid,        comprising:        -   a first chamber including an agitator for agitating the            fluid;        -   a second, outer chamber including at least one bed for            culturing cells; and        -   a third, inner chamber for returning fluid from the second            outer chamber to the first chamber,        -   wherein the second, outer chamber is bounded by an outer            wall, and further including an outer casing forming a space            with the outer wall, the space being for insulating,            heating, or cooling the second, outer chamber.    -   60. The bioreactor of item 59, wherein the at least one bed        comprises a structured fixed bed.    -   61. The bioreactor of item 58 or item 59, wherein the structured        fixed bed comprises a spiral bed.    -   62. The bioreactor of item 59, wherein the inner chamber is        formed by at least one tube.    -   63. The bioreactor of item 62, wherein the at least one tube is        connected to first and second supports bounding the at least one        bed.    -   64. The bioreactor of item 63, wherein the first and second        supports are connected to the outer wall.    -   65. The bioreactor of item 63 or 64, wherein the first and        second supports are at least partially perforated.    -   66. An apparatus for culturing cells, comprising: a bioreactor        including an agitator, the bioreactor adapted for maintaining        the agitator in a suspended condition that allows side-to-side        movement for alignment with an external drive (which suspended        agitator may form part of any of items 1-65).    -   67. The apparatus of item 66, wherein the bioreactor includes a        base portion for receiving the agitator, and an intermediate        portion for supporting a carrier for carrying the agitator in        the suspended condition.    -   68. The apparatus of item 67, wherein the carrier comprises a        clip for engaging the intermediate portion.    -   69. An apparatus for culturing cells, comprising:        -   a bioreactor including an agitator having a plurality of            curved blades (which agitator may form part of any of items            1-68).    -   70. The apparatus of item 69, wherein the agitator includes a        central open region radially inward of the plurality of curved        blades.    -   71. The apparatus of item 69 or item 70, wherein the agitator        includes one or more magnets.    -   72. The apparatus of any of items 69-71, wherein the blades are        curved in a radial direction.    -   73. A bioreactor comprising first and second stacked, structured        beds.    -   74. The bioreactor of item 73, further including a screen        engaging both the first and second stacked, structured beds.    -   75. The bioreactor of item 73 or item 74, wherein the first and        second stacked, structured beds comprise spiral beds.    -   76. A bioreactor including a structured fixed bed forming a        central column of the bioreactor.    -   77. The bioreactor of item 76, wherein the structured fixed bed        comprises a spiral bed.    -   78. The bioreactor of item 76 or item 77, wherein an inner        surface of the structured fixed bed is fluid-impervious.    -   79. A method of manufacturing a bioreactor, comprising:        -   connecting a base portion including a first chamber with an            agitator for agitating a fluid to at least one intermediate            portion forming at least portion of a second, outer chamber            for culturing cells in connection with fluid transferred            from the second, outer chamber, and a third, inner chamber            for returning fluid to the first chamber of the base            portion.    -   80. The method of item 79, further including the step of        connecting a cover portion over the at least one intermediate        portion.    -   81. The method of item 79 or item 80, further including the step        of spirally wrapping a matrix material around the intermediate        portion to form a structured fixed bed for culturing cells in        the outer chamber.    -   82. The method of any of items 79-81, further including the step        of inserting a structured fixed bed into the second, outer        chamber.    -   83. The method of any of items 79-82, further including        providing an outer casing to form a periphery of the second,        outer chamber.    -   84. The method of any of items 79-83, further including the step        of connecting the outer casing to the base portion.    -   85. The method of any of items 79-84, further including the step        of stacking a plurality of structured fixed beds in the second,        outer chamber.    -   86. The method of any of items 79-85, further including the step        of providing a perforated support between the plurality of        structured fixed beds.    -   87. The method of any of items 79-86 further including the step        of suspending the agitator above the base portion in a manner        that permits side-to-side movement to align with an external        drive.    -   88. A method of manufacturing a bioreactor, comprising:        -   providing a plurality of structured fixed beds in the            bioreactor.    -   89. The method of item 88, further including the step of        providing a perforated spacer between each of the plurality of        structured fixed beds.    -   90. The method of item 88 or 89, further including the step of        providing an inner tube along an inner side of each structured        fixed bed and an outer tube along an outer side of each        structured fixed bed.    -   91. The method of any of items 88-90, further including the step        of providing a casing radially outward of the outer tube, the        casing creating a space for insulating, heating, or cooling the        bioreactor.

As used herein, the following terms have the following meanings:

“A”, “an”, and “the” as used herein refers to both singular and pluralreferents unless the context clearly dictates otherwise. By way ofexample, “a compartment” refers to one or more than one compartment.

“About,” “substantially,” or “approximately,” as used herein referringto a measurable value, such as a parameter, an amount, a temporalduration, and the like, is meant to encompass variations of +/−20% orless, preferably +/−10% or less, more preferably +/−5% or less, evenmore 35 preferably +/−1% or less, and still more preferably +/−0.1% orless of and from the specified value, in so far such variations areappropriate to perform in the disclosed invention. However, it is to beunderstood that the value to which the modifier “about” refers is itselfalso specifically disclosed.

“Comprise”, “comprising”, and “comprises” and “comprised of” as usedherein are synonymous with “include”, “including”, “includes” or“contain”, “containing”, “contains” and are inclusive or open-endedterms that specifies the presence of what follows e.g. component and donot exclude or preclude the presence of additional, non-recitedcomponents, features, element, members, steps, known in the art ordisclosed therein.

While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the invention. For example, while thebioreactor is shown in a vertical orientation, it could be used in anyorientation. It should be understood that various alternatives to theembodiments of the invention described herein may be employed inpracticing the invention. It is intended that the following claimsdefine the scope of the protection under the applicable law and thatmethods and structures within the scope of these claims and theirequivalents be covered thereby.

1. An apparatus for culturing cells in connection with a fluid flow,comprising: a bioreactor including a structured fixed bed for culturingcells, the bioreactor comprising a housing forming an interiorcompartment, the interior compartment including: a first chambercomprising the structured fixed bed for culturing cells; and a secondchamber within the first chamber, the second chamber defined by an outerwall comprising an imperforate tube for transmitting fluid having passedthrough the structured fixed bed back to the first chamber, wherein thestructured fixed bed comprises a plurality of layers of materialspirally wound around the imperforate tube.
 2. The apparatus accordingto claim 1, the interior compartment further including a third chamberfor recirculating fluid through the structured fixed bed.
 3. Theapparatus according to claim 2, wherein the third chamber is below thefirst chamber and includes an impeller.
 4. The apparatus according toclaim 2, wherein the third chamber is above the first chamber.
 5. Theapparatus according to claim 3, wherein the impeller comprises one ormore curved blades.
 6. The apparatus according to claim 1, furtherincluding an external reservoir and conduits forming a continuous loopfor circulating fluid to the bioreactor.
 7. The apparatus according toclaim 1, wherein the plurality of layers of material comprise a meshlayer.
 8. The apparatus according to claim 1, wherein the plurality oflayers of material comprise a woven material.
 9. The apparatus accordingto claim 8, wherein the plurality of layers of woven material are indirect contact.
 10. An apparatus for culturing cells in connection witha fluid flow, comprising: a bioreactor including a structured fixed bedfor culturing cells, the bioreactor comprising a housing forming aninterior compartment, the interior compartment including a first chambercomprising the structured fixed bed for culturing cells and a secondchamber within the first chamber, the second chamber defined by an outerwall forming an imperforate tube for transmitting fluid having passedthrough the structured fixed bed back to the first chamber, wherein thestructured fixed bed comprises a plurality of layers of material. 11.The apparatus according to claim 10, further including an externalreservoir and conduits for circulating fluid to the bioreactor.
 12. Theapparatus according to claim 10, wherein the plurality of layers ofmaterial comprise one or more woven layers.
 13. The apparatus accordingto claim 10, wherein the first chamber comprises a top wall having anopening for transmitting fluid from the first chamber to the secondchamber.
 14. The apparatus according to claim 10, wherein the firstchamber is bounded by an outer wall, and further including an outercasing forming a space with the outer wall.
 15. The apparatus accordingto claim 10, wherein the structured fixed bed comprises a spirally woundfixed bed.
 16. The apparatus according to claim 10, wherein theplurality of layers of materials comprise cell immobilization layers indirect contact.
 17. An apparatus for culturing cells in connection witha fluid flow, comprising: a modular bioreactor including a structuredfixed bed for culturing cells, the bioreactor comprising a housingforming an interior compartment, the interior compartment including thestructured fixed bed for culturing cells, wherein the structured fixedbed comprises a plurality of layers of woven material in direct contactwith one another; and an external reservoir and conduits for circulatingfluid to the bioreactor.
 18. The apparatus according to claim 17,wherein the plurality of layers of woven material are spirally wouldaround an imperforate tube.
 19. The apparatus according to claim 17,wherein the plurality of layers of woven material are in direct contact.20. The apparatus according to claim 17, wherein the conduits areconnected to ports in a lid of the bioreactor.